RNA viruses cause a variety of emerging and biodefense-related diseases of public health importance. The viruses come from multiple families, and the diseases they cause are diverse. They include West Nile encephalitis and dengue fever both caused by flaviviruses, Venezuelan equine encephalitis and chikungunya caused by alphaviruses and Rift Valley fever caused by a bunyavirus. Current therapies for these diseases are limited, and there is a real need to develop effective small molecule antiviral agents. The central objectives of this five year project are to meet this need by identifying and developing new antivirals with activity against medically important RNA viruses giving particular emphasis to broadly active candidates. In aim 1 we will develop compounds active against flaviviruses from diverse chemical leads identified by high throughput screening (HTS) using a West Nile virus virus-replicon particle (VRP) assay that can be used under low biocontainment conditions. The compounds will undergo an iterative program with multiple rounds of chemical modifications, and in vitro toxicity and antiviral activity testing against flaviviruses that will result in the identification of improved antivirals. In aim 2 we will develop an alphavirus VRP assay into a HTS-suitable format and use this assay to screen for lead compounds that will then be used to initiate an alphavirus antiviral discovery and development program using the paradigm described in aim 1. In aim 3 we will develop an HTS assay that utilizes an attenuated Rift Valley fever virus that expresses a reporter gene to initiate a bunyavirus antiviral program. In each aim activity will be evaluated using multiple viruses within the family, to identify compounds with broad activity. This process will be continued by evaluating active compounds against members of the other virus families of interest. In doing this we recognize that the early clinical signs caused by many of these viruses are difficult to distinguish;thus, broad spectrum antivirals that could be used early in infection when they would have the best chance of clinical impact are a clinical priority. Concomitant with the drug discovery studies, in aim 4 we will explore the mechanism of action of compounds, generate resistant mutations to define the frequency with which they occur, and determine the mechanism of resistance. In addition, we will explore the effects of using active compounds in combination, in attempts to identify synergistic combinations with potential future clinical utility. Finally in aim 5 we will evaluate two promising compounds in vivo in mouse models of flavivirus diseases.